The long-term aim of this proposal is to identify the molecular components that regulate exocytotic fusion in the mast cell, and determine their mode of action. It is known that exocytosis is regulated by Ca++ and guanine nucleotides. However outstanding questions remain: what is the role of spatially localized calcium in regulating exocytosis; what is the nature of the calcium and guanine nucleotide binding proteins; what structural proteins are targets for these regulatory proteins; and, how does the assembled fusion machinery control membrane fusion? In this proposal we will study the regulation of exocytosis using a combination of biophysical, biochemical and molecular biology techniques. Exocytosis will be measured in single cells with a patch-clamp assay that can detect the fusion of single secretory granules, while controlling the composition of the cytosol. Photolabile 'caged' compounds will be used to create well defined intracellular stimuli and laser controlled imaging techniques will be used to measure sharp intracellular Ca++ gradients. Further manipulations will involve cytosolic perfusion with synthetic peptides, anti-peptide antibodies and intranuclear injection of antisense oligonucleotides to block the expression of specific genes. We will use these techniques to: 1) directly test the hypothesis that a sharp and large Ca++ gradient beneath the plasma membrane is sufficient to cause exocytosis; 2) investigate the role of putative structural and regulatory proteins such as Annexins (Ca++ binding), heterotrimeric GTP binding proteins, low molecular weight G proteins (e.g., Rab3) and Secretory Carrier Membrane Proteins (SCAMP's), in the control of the final molecular events that cause exocytotic fusion; and 3) to develop an in vitro assay of fusion using isolated intact patch clamped secretory granules to directly observe the activity of granule membrane proteins and to monitor the fusion of patch-clamped granules with lipid vesicles under conditions that simulate the cytosol. This assay will be developed as a tool for testing, in vitro, specific mechanistic models that describe the relationship between intracellular messengers, regulatory proteins and the formation of the fusion pore.